The disclosed processes relate generally to the field of genomics, proteomics and molecular medicine, and more specifically to processes of using infrared matrix assisted laser desorption-ionization mass spectrometry to analyze, or otherwise detect the presence of or determine the identity of a biological macromolecule.
In recent years, the molecular biology of a number of human genetic diseases has been elucidated by the application of recombinant DNA technology. More than 3000 diseases are known to be of genetic origin (Cooper and Krawczak, xe2x80x9cHuman Genome Mutationsxe2x80x9d (BIOS Publ. 1993)), including, for example, hemophilias, thalassemias, Duchenne muscular dystrophy, Huntington""s disease, Alzheimer""s disease and cystic fibrosis, as well as various cancers such as breast cancer. In addition to mutated genes that result in genetic disease, certain birth defects are the result of chromosomal abnormalities, including, for example, trisomy 21 (Down""s syndrome), trisomy 13 (Patau syndrome), trisomy 18 (Edward""s syndrome), monosomy X (Turner""s syndrome) and other sex chromosome aneuploidies such as Klinefelter""s syndrome (XXY).
Other genetic diseases are caused by an abnormal number of trinucleotide repeats in a gene. These diseases include Huntington""s disease, prostate cancer, spinal cerebellar ataxia 1 (SCA-1), Fragile X syndrome (Kremer et al., Science 252:1711-14 (1991); Fu et al., Cell 67:1047-58 (1991); Hirst et al., J. Med. Genet. 28:824-29 (1991)); myotonic dystrophy type I (Mahadevan et al., Science 255:1253-55 (1992); Brook et al., Cell 68:799-808 (1992)), Kennedy""s disease (also termed spinal and bulbar muscular atrophy (La Spada et al., Nature 352:77-79 (1991)), Machado-Joseph disease, and dentatorubral and pallidolyusian atrophy. The aberrant number of triplet repeats can be located in any region of a gene, including a coding region, a non-coding region of an exon, an intron, or a regulatory element such as a promoter. In certain of these diseases, for example, prostate cancer, the number of triplet repeats is positively correlated with prognosis of the disease.
Evidence indicates that amplification of a trinucleotide repeat is involved in the molecular pathology in each of the disorders listed above. Although some of these trinucleotide repeats appear to be in non-coding DNA, they clearly are involved with perturbations of genomic regions that ultimately affect gene expression. Perturbations of various dinucleotide and trinucleotide repeats resulting from somatic mutation in tumor cells also can affect gene expression or gene regulation.
Additional evidence indicates that certain DNA sequences predispose an individual to a number of other diseases, including diabetes, arteriosclerosis, obesity, various autoimmune diseases and cancers such as colorectal, breast, ovarian and lung cancer. Knowledge of the genetic lesion causing or contributing to a genetic disease allows one to predict whether a person has or is at risk of developing the disease or condition and also, at least in some cases, to determine the prognosis of the disease.
Numerous genes have polymorphic regions. Since individuals have any one of several allelic variants of a polymorphic region, each can be identified based on the type of allelic variants of polymorphic regions of genes. Such identification can be used, for example, for forensic purposes. In other situations, it is crucial to know the identity of allelic variants in an individual. For example, allelic differences in certain genes such as the major histocompatibility complex (MHC) genes are involved in graft rejection or graft versus host disease in bone marrow transplantation. Accordingly, it is highly desirable to develop rapid, sensitive, and accurate methods for determining the identity of allelic variants of polymorphic regions of genes or genetic lesions.
Several methods are used for identifying allelic variants or genetic lesions. For example, the identity of an allelic variant or the presence of a genetic lesion can be determined by comparing the mobility of an amplified nucleic acid fragment with a known standard by gel electrophoresis, or by hybridization with a probe that is complementary to the sequence to be identified. Identification only can be accomplished, however, if the nucleic acid fragment is labeled with a sensitive reporter function, for example, a radioactive (32P, 35S), fluorescent or chemiluminescent reporter. Radioactive labels can be hazardous and the signals they produce can decay substantially over time. Non-radioactive labels such as fluorescent labels can suffer from a lack of sensitivity and fading of the signal when high intensity lasers are used. Additionally, labeling, electrophoresis and subsequent detection are laborious, time-consuming and error-prone procedures. Electrophoresis is particularly error-prone, since the size or the molecular weight of the nucleic acid cannot be correlated directly to its mobility in the gel matrix because sequence specific effects, secondary structures and interactions with the gel matrix cause artifacts in its migration through the gel.
Applications of mass spectrometry in the biosciences have been reported (see Meth. Enzymol., Vol. 193, Mass Spectrometry (McCloskey, ed.; Academic Press, NY 1990); McLaffery et al., Acc. Chem. Res. 27:297-386 (1994); Chait and Kent, Science 257:1885-1894 (1992); Siuzdak, Proc. Natl. Acad. Sci., USA 91:11290-11297 (1994)), including methods for mass spectrometric analysis of biopolymers (see Hillenkamp et al. (1991) Anal. Chem. 63:1193A-1202A) and for producing and analyzing biopolymer ladders (see, International Publ. WO 96/36732; U.S. Pat. No. 5,792,664).
Mass spectrometry has been used for the analysis of nucleic acids (see, for example, Schram, Mass Spectrometry of Nucleic Acid Components, Biomedical Applications of Mass Spectrometry 34:203-287 (1990); Crain, Mass Spectrom. Rev. 9:505-554 (1990); Murray, J. Mass Spectrom. Rev. 31:1203 (1996); Nordhoff et al., Mass Spectrom. Rev. 15:67-138 (1997); U.S. Pat. No. 5,547,835; U.S. Pat. No. 5,605,798; PCT Application Publication No. W094/16101; PCT Application Publication No. WO 96/29431).
The so-called xe2x80x9csoft ionizationxe2x80x9d mass spectrometric methods, including Matrix-Assisted Laser Desorption/Ionization (MALDI) and ElectroSpray Ionization (ESI), allow intact ionization, detection and mass determination of large molecules, i.e., well exceeding 300 kDa in mass (Fenn et al., Science 246:64-71 (1989); Karas and Hillenkamp, Anal. Chem. 60:2299-3001 (1988)). MALDI mass spectrometry (MALDI-MS; reviewed in Nordhoff et al., Mass Spectrom. Rev. 15:67-138 (1997)) and ESI-MS have been used to analyze nucleic acids. Nucleic acids are very polar biomolecules that are difficult to volatilize and, therefore, there has been an upper mass limit for clear and accurate resolution.
ESI has been used for the intact desorption of large nucleic acids even in the megaDalton mass range (Ferstenau and Benner, Rapid Commun. Mass Spectrom. 9:1528-1538 (1995); Chen et al., Anal. Chem. 67:1159-1163 (1995)). Mass assignment using ESI is very poor and only possible with an uncertainty of about 10%. The largest nucleic acids that have been accurately mass determined by ESI-MS are a 114 base pair double stranded PCR product (Muddiman et al., Anal. Chem. 68:3705-3712 (1996)) of about 65 kDA in mass and a 120 nucleotide E.coli 5S rRNA of about 39 kDa in mass (Limbach et al., J. Am. Soc. Mass Spectrom. 6:27-39 (1995)). Furthermore, ESI requires extensive sample purification.
MALDI-MS requires incorporation of the macromolecule to be analyzed in a matrix, and has been performed on polypeptides and on nucleic acids mixed in a solid (i.e., crystalline) matrix. In these methods, a laser is used to strike the biopolymer/matrix mixture, which is crystallized on a probe tip, thereby effecting desorption and ionization of the biopolymer. In addition, MALDI-MS has been performed on polypeptides using the water of hydration (i.e., ice) or glycerol as a matrix. When the water of hydration was used as a matrix, it was necessary to first lyophilize or air dry the protein prior to performing MALDI-MS (Berkenkamp et al. (1996) Proc. Natl. Acad. Sci. USA 93:7003-7007). The upper mass limit for this method was reported to be 30 kDa with limited sensitivity (i.e., at least 10 pmol of protein was required). Infrared MALDI-MS of proteins reportedly consumes 100-1000 times more material per spectrum as compared to UV MALDI-MS and, in combination with matrices such as glycerol, can tend to form adducts which broaden the peaks on the high mass side (Hillenkamp et al. (1995) 43rd ASMS Conference on Mass Spectrometry and Allied Topics, p. 357). Furthermore, although IR-MALDI MS appeared to provide increased mass resolution due to less metastable fragmentation as compared to UV-MALDI MS, this decrease in metastable decay has been reported to be accompanied by an increase in fragmentation.
UV-MALDI-MS is limited in the size of biological macromolecules that can be analyzed. For example, it is difficult to analyze nucleic acid molecules much larger than about 100 nucleotides (100-mer) by UV-MALDI-MS.
Accordingly, despite the effort to apply mass spectrometry methods to the analysis of nucleic acid molecules, limitations remain due, in part, to physical and chemical properties of nucleic acids. For example, the polar nature of nucleic acid biopolymers makes them difficult to volatilize.
Analysis of large DNA molecules using UV-MALDI-MS has been reported (Ross and Belgrader, Anal. Chem. 69:3966-3972 (1997); Tang et al., Rapid Commun. Mass Spectrom. 8:727-730 (1994); Bai et al., Rapid Commun. Mass Spectrom. 9:1172-1176 (1995); Liu et al., Anal. Chem. 67:3482-3490 (1995); Siegert et al., Anal. Biochem. 243:55-65 (1997)). Based on these reports, it is clear that analysis of nucleic acids exceeding 30 kDa in mass (approximately a 100-mer) by UV-MALDI-MS becomes increasingly difficult with a current upper mass limit of about 90 kDa (Ross and Belgrader, Anal. Chem. 69:3966-3972 (1997)). The inferior quality of the DNA UV-MALDI spectra has been attributed to a combination of ion fragmentation and multiple salt formation of the phosphate backbone. Since RNA is considerably more stable than DNA under UV-MALDI conditions, the accessible mass range for RNA is up to about 150 kDa (Kirpekar et al., Nucl. Acids Res. 22:3866-3870 (1994)).
Nucleic acids in solid matrices (mostly succinic acid and, to a lesser extent, urea and nicotinic acid) have been analyzed by IR-MALDI (Nordhoff et al., Rapid Commun. Mass Spectrom. 6:771-776 (1992); Nordhoff et al., Nucl. Acids Res. 21: 3347-3357 (1993); Nordhoff et al., J. Mass Spec. 30:99-112 (1995)). Nordhoff et al. (1992) initially reported that a 20-mer of DNA and an 80-mer of RNA were about the uppermost limit for resolution. Nordhoff et al. (1993) later provided distinct spectra for a 26-mer of DNA and a 104-mer of tRNA and reported that reproducible signals were obtained for RNA up to 142 nucleotides. Nordhoff et al. (1995) also reported a substantially better spectra for the analysis of a 40-mer by UV-MALDI with the solid matrix, 3-hydroxy picolinic acid, than by IR-MALDI with succinic acid, but that IR-MALDI resulted in a substantial degree of prompt fragmentation.
Analysis of macromolecules in a biological sample, for example, can provide information as to the condition of the individual from which the sample was obtained. For example, nucleic acid analysis of a biological sample obtained from an individual can be useful for diagnosing the existence of a genetic disease or chromosomal abnormality, a predisposition to a disease or condition, or an infection by a pathogenic organism, or can provide information relating to identity, heredity or compatibility. Since mass spectrometry can be performed relatively quickly and is amenable to automation, improved methods for obtaining accurate mass spectra for biological macromolecules, particularly for larger nucleic acid molecules larger than about 90 kDa for DNA and 150 kDA for RNA are needed.
Accordingly, a need exists for methods to detect and characterize biological macromolecules such as nucleic acid molecules, including methods to detect genetic lesions in a nucleic acid molecule. There is a need for accurate, sensitive, precise and reliable methods for detecting and characterizing biological macromolecules, particularly in connection with the diagnosis of conditions, diseases and disorders. Therefore it is an object herein to provide processes that satisfy these needs and provide additional advantages.
Processes for the determination of the mass or identity of biological macromolecules using infrared matrix assisted laser desorption/ionization (IR-MALDI) mass spectrometry and a liquid matrix are provided. In particular, infrared matrix assisted laser desorption/ionization (IR-MALDI) mass spectrometry of nucleic acids, including DNA and RNA, in a liquid matrix are provided. The liquid matrix (liquid at room temperature, one atmosphere pressure) is an IR-absorbing biocompatible material, such as a polyglycol, particularly glycerol, that can form a glass or vitreous solid. The use of IR-MALDI and this liquid matrix can be employed in any method, particularly diagnostic methods and sequencing methods, heretofore performed with UV-MALDI. Such methods, particularly diagnostic methods for nucleic acids and proteins, include, but are not limited to, those described in U.S. Pat. Nos. 5,547,835, 5,691,141, 5,605,798, 5,622,824, 5,777,324, 5,830,655, 5,700,642, allowed U.S. application Ser. Nos. 08/617,256, 08/746,036, 08/744,481, 08/744,590, 08/647,368, published International PCT application Nos. WO 96/29431, WO 99/12040, WO 98/20019, WO 98/20166, WO 98/20020, WO 97/37041, WO 99/14375, WO 97/42348, WO 98/54751 and WO 98/26095.
In practicing an embodiment of the method for nucleic acid analyses, a composition for IR-MALDI containing the nucleic acid and a liquid matrix is deposited onto a substrate, which, generally, is a solid support, to form a homogeneous, transparent thin layer of nucleic acid mixture. This mixture is illuminated with infrared radiation so that the nucleic acid solution is desorbed and ionized, thereby emitting ion particles, which are analyzed using a mass analyzer to determine the mass of the nucleic acid. Preferably, sample preparation and deposition are performed using an automated device.
Methods for detecting the presence or absence of a biological macromolecule in a sample using IR-MALDI mass spectrometry are also provided herein. In a particular embodiment, a composition for IR-MALDI containing the biological macromolecule and a matrix is illuminated with infrared radiation, desorbed and ionized, thereby emitting ion particles, which are analyzed to determine whether the nucleic acid is present.
Methods for detecting the presence or absence of a nucleic acid in a sample using IR-MALDI mass spectrometry are also provided herein. In a particular embodiment, a composition for IR-MALDI containing the sample and a liquid matrix is illuminated with infrared radiation, desorbed and ionized, thereby emitting ion particles which are analyzed to determine whether the nucleic acid is present.
Liquid matrices for use in the processes disclosed herein have a sufficient absorption at the wavelength of the laser to be used in performing desorption and ionization and are a liquid at room temperature (20xc2x0 C.) and can form a vitreous or glass solid. The liquid is intended to be used in any IR MALDI format and at any temperature, typically about xe2x88x92200xc2x0 C. to 80xc2x0 C., preferably xe2x88x9260xc2x0 C. to about 40xc2x0 C., suitable for such formats.
For absorption purposes, the liquid matrix can contain at least one chromophore or functional group that strongly absorbs infrared radiation. Preferred functional groups include nitro, sulfonyl, sulfonic acid, sulfonamide, nitrile or cyanide, carbonyl, aldehyde, carboxylic acid, amide, ester, anhydride, ketone, amine, hydroxyl, aromatic rings, dienes and other conjugated systems.
Among the preferred liquid matrices are substituted or unsubstituted (1) alcohols, including glycerol, sugars, polysaccharides, 1,2-propanediol, 1,3-propanediol, 1,2-butanediol, 1,3-butanediol, 1,4-butanediol and triethanolamine; (2) carboxylic acids, including formic acid, lactic acid, acetic acid, propionic acid, butanoic acid, pentanoic acid and hexanoic acid, or esters thereof; (3) primary or secondary amides, including acetamide, propanamide, butanamide, pentanamide and hexanamide, whether branched or unbranched; (4) primary or secondary amines, including propylamine, butylamine, pentylamine, hexylamine, heptylamine, diethylamine and dipropylamine; and (5) nitriles, hydrazine and hydrazide. The liquids do not crystallize, but rather can form a glass or vitreous phase when subjected to drying, cooling or other conditions leading to a transition from the liquid phase. Materials of relatively low volatility are preferred to avoid rapid evaporation under conditions of vacuum during the IR-MALDI processes.
Preferably, a liquid matrix for use herein is miscible with a nucleic acid compatible solvent. As noted, it is also preferable that the liquid matrix is vacuum stable, i.e., has a low vapor pressure, so that the sample does not evaporate quickly in the mass analyzer. Preferably the liquid has an appropriate viscosity to facilitate dispensing of microliter to nanoliter volumes of matrix, either alone or mixed with a nucleic acid compatible solvent. Mixtures of different liquid matrices and additives to such matrices may be desirable to confer one or more of the properties described above. Such mixtures can contain two liquid matrix materials (i.e.,. binary mixtures), three (tertiary mixtures) or more.
A nucleic acid/matrix composition for IR-MALDI is deposited as a thin layer on a substrate, which preferably is contained with a vacuum chamber. Preferred substrates for holding the nucleic acid/matrix solution can be solid supports, for example, beads, capillaries, flat supports, pins or wafers, with or without filter plates. Preferably the temperature of the substrate can be regulated to cool the nucleic acid/matrix composition to a temperature that is below room temperature.
Preferred infrared radiation is in the mid-IR wavelength region from about 2.5 xcexcm to about 12 xcexcm. Particularly preferred sources of radiation include CO, CO2 and Er lasers. In certain embodiments, the laser can be an optic fiber laser, or the laser radiation can be coupled to the mass spectrometer by fiber optics.
In a further preferred embodiment, the ion particles generated by infrared irradiation of the analyte in the liquid matrix are extracted for analysis by the mass analyzer in a delayed fashion prior to separation and detection in a mass analyzer. Preferred separation formats include linear or reflector, with linear and nonlinear fields, for example, curved field reflectron; time-of-flight (TOF); single or multiple quadrupole; single or multiple magnetic sector; Fourier transform ion cyclotron resonance (FTICR); or ion trap mass spectrometers.
Processes of using IR-MALDI mass spectrometry to identify the presence of a target nucleic acid in a biological sample are provided. Such a process can be performed, for example, by amplifying nucleic acid molecules in the biological sample; contacting the amplified nucleic acid molecules with a detector oligonucleotide, which can hybridize to a target nucleic acid sequence present among the amplified nucleic acid molecules; preparing a composition for IR-MALDI, by mixing the product of the reaction with a liquid matrix, which absorbs infrared radiation; and
identifying duplex nucleic acid molecules in the composition by IR-MALDI mass spectrometry, wherein the presence of duplex nucleic acid molecules identifies the presence of the target nucleic acid in the biological sample.
A process for identifying the presence of a target nucleic acid sequence in a biological sample also can be performed by amplifying nucleic acid molecules obtained from a biological sample; specifically digesting the amplified nucleic acid molecules using at least one appropriate nuclease, to produce digested fragments; hybridizing the digested fragments with complementary capture nucleic acid sequences, which are immobilized on a solid support and can hybridize to a digested fragment of a target nucleic acid to produce immobilized fragments; preparing a composition for IR-MALDI, containing the immobilized fragments and a liquid matrix, which absorbs infrared radiation; and identifying immobilized fragments by IR-MALDI mass spectrometry, thereby detecting the presence of the target nucleic acid sequence in the biological sample.
The presence of a target nucleic acid in a biological sample also can be identified by performing on nucleic acid molecules obtained from the biological sample, a first polymerase chain reaction using a first set of primers, which are capable of amplifying a portion of the nucleic acid containing the target nucleic acid; preparing a composition containing the first amplification product and a liquid matrix, which absorbs infrared radiation; and detecting the first amplification product in the composition by IR-MALDI mass spectrometry, thereby detecting the presence of the target nucleic acid in the biological sample. If desired, such a process can include, prior to preparing the composition for IR-MALDI, performing a second polymerase chain reaction on the first amplification product using a second set of primers that can amplify at least a portion of the first amplification product containing the target nucleic acid.
Also disclosed herein are compositions, particularly compositions for IR-MALDI, such compositions containing a biological macromolecule, which is suitable for analysis by IR-MALDI, and a liquid matrix, which absorbs infrared radiation. A biological macromolecule suitable for analysis by IR-MALDI can be, for example, a nucleic acid, a polypeptide or a carbohydrate, or can be a macromolecular complex such as a nucleoprotein complex, protein-protein complex, or the like. A composition for IR-MALDI as disclosed herein generally contains the biological macromolecule, for example, a nucleic acid, and the liquid matrix in a ratio of about 10xe2x88x924 to 10xe2x88x929, and can contain less than about 10 picomoles of biological macromolecule to be analyzed, for example, about 100 attomol to about 1 picomole (pmol) of the biological macromolecule. (For proteins, the analyte to matrix ratio is typically narrower ranging from about 2xc3x9710xe2x88x924 to 2xc3x9710xe2x88x925). A composition for IR-MALDI as disclosed herein also can contain an additive, which facilitates detection of the biological macromolecule by IR-MALDI, for example, an additive that improves the miscibility of the biological macromolecule in the liquid matrix. In one embodiment, a composition for IR-MALDI is deposited on a substrate, which can be a solid support such as a silicon wafer or other material providing a surface for deposition of a composition for IR-MALDI, for example, a stainless steel surface.
Processes for characterizing a biological macromolecule by IR-MALDI mass spectrometry are provided. For example, the mass of a biological macromolecule can be determined by preparing a composition for IR-MALDI containing the biological macromolecule to be analyzed and a liquid matrix, which absorbs infrared radiation; then analyzing the biological macromolecule in the composition by IR-MALDI mass spectrometry, thereby allowing a determination of the mass of the biological macromolecule.
A process as disclosed herein also can be used for detecting a target biological macromolecule by preparing a composition for IR-MALDI containing the target biological macromolecule and a liquid matrix, which absorbs infrared radiation, and performing IR-MALDI mass spectrometry on the composition to identify the target biological macromolecule in the composition, thereby detecting the target biological macromolecule. If desired, the target biological macromolecule can be present in or obtained from a biological sample. Accordingly, a process for identifying the presence of a target biological macromolecule in a biological sample, is provided. The presence of a target nucleic acid, for example, can be identified by preparing a composition for IR-MALDI, containing a biological sample containing nucleic acid molecules (or nucleic acid molecules isolated from the biological sample) and a liquid matrix, which absorbs infrared radiation; then analyzing the composition by IR-MALDI mass spectrometry, wherein detection of a nucleic acid molecule having a molecular mass of the target nucleic acid sequence identifies the presence of the target nucleic acid sequence in the biological sample.
Also provided is a process of using IR-MALDI mass spectrometry to identify an individual having a disease or a predisposition to a disease by detecting a characteristic of a biological macromolecule that is obtained from the individual and is associated with the disease or the predisposition. Such a process is particularly useful for identifying a genetic disease, or a disease associated with a bacterial infection, or a predisposition to such a disease, and also is useful for determining identity, heredity or compatibility.
The processes disclosed herein are suitable for analyzing one or more target biological macromolecules, particularly a large number of target biological macromolecules, for example, by depositing a plurality of compositions, each containing one or more target biological macromolecules, on a solid support, for example, a chip, in the form of an array. The disclosed processes are particularly suitable for multiplex analysis of a plurality of biological macromolecules contained in a single composition, including a liquid matrix, in which case each biological macromolecule in the plurality can be differentially mass modified to facilitate multiplex analysis. Accordingly, the processes disclosed herein are readily adaptable to high throughput assay formats.
Processes for obtaining information on a sequence of a nucleic acid molecule by determining the identity of a target polypeptide encoded by the nucleic acid molecule are provided. In practicing these methods, a target polypeptide (or mixture thereof) is prepared from a nucleic acid molecule encoding the target polypeptide; the molecular mass of the target polypeptide is determined by providing a mixture of the polypeptide with a liquid matrix, or in some embodiments, with water or succinic acid, and preforming IR-MALDI. The identity of the target polypeptide is determined by comparing the molecular mass of the target polypeptide with the molecular mass of a reference polypeptide of known identity. Information, such as the presence of a mutation, on a sequence of nucleotides in the nucleic acid molecule encoding the target polypeptide can thereby be obtained.
A biological macromolecule particularly suitable for analysis by a process of IR-MALDI mass spectrometry can be a nucleic acid, a nucleic acid analog or mimic, a triple helix, a polypeptide, a polypeptide analog or mimetic, a carbohydrate, a lipid or a proteoglycan, or can be a macromolecular complex such as a protein-protein complex or a nucleoprotein complex or other complexes. For analysis by a process as disclosed herein, a target biological macromolecule can be immobilized to a substrate, particularly a solid support, which can be, for example, a bead, a flat surface, a chip, a capillary, a pin, a comb, or a wafer, and can be any of various materials, including a metal, a ceramic, a plastic, a resin, a gel, and a membrane. Immobilization can be through a reversible linkage (i.e. an ionic bond, such as biotin/streptavidin), a covalent bond, such as a photocleavable bond or a thiol linkage or a hydrogen bond, and the linkage can be cleaved using, for example, a chemical process, an enzymatic process, or a physical process, including the IR-MALDI mass spectrometric analysis procedure.
A biological macromolecule to be analyzed can be conditioned prior to IR-MALDI mass spectrometric analysis, thereby improving the ability to analyze the particular biological macromolecule by IR-MALDI mass spectrometry, for example, by improving the resolution of the mass spectrum. A target biological macromolecule can be conditioned, for example, by ion exchange, by contact with an alkylating agent or trialkylsilyl chloride, or by incorporation of at least one mass modified subunit of the biological macromolecule. If desired, the biological macromolecule can be isolated prior to conditioning or prior to IR-MALDI mass spectrometric analysis.
A process for determining the identity of each target biological macromolecule in a plurality of target biological macromolecules, which can be fragments of a biological macromolecule, can be performed, for example, by preparing a composition for IR-MALDI containing a plurality of differentially mass modified target biological macromolecules and a liquid matrix, which absorbs infrared radiation; determining the molecular mass of each differentially mass modified target biological macromolecule in the plurality by IR-MALDI mass spectrometry; and comparing the molecular mass of each differentially mass modified target biological macromolecule in the plurality with the molecular mass of a corresponding known biological macromolecule. Where such a process is performed using a plurality of target biological macromolecules, each of which is a fragment of a larger biological macromolecule, the fragments can be prepared by contacting the biological macromolecules with at least one agent that cleaves a bond involved in the formation of the biological macromolecules, particularly a bond between monomer subunits of the biological macromolecule.
Processes for identifying one or more subunits in a biological macromolecule using IR-MALDI mass spectrometry also are provided, for example, processes for detecting a mutation in a nucleotide sequence. The identity of a target nucleotide can be identified, for example, by hybridizing a nucleic acid molecule containing the target nucleotide with a primer oligonucleotide that is complementary to the nucleic acid molecule at a site adjacent to the target nucleotide, to produce a hybridized nucleic acid molecule; contacting the hybridized nucleic acid molecule with a complete set of dideoxynucleosides or 3xe2x80x2-deoxynucleoside triphosphates and a DNA dependent DNA polymerase, so that only the dideoxynucleosides or 3xe2x80x2-deoxynucleoside triphosphate that is complementary to the target nucleotide is extended onto the primer; preparing a composition containing the extended primer and a liquid matrix, which absorbs infrared radiation; and detecting the extended primer in the composition by IR-MALDI mass spectrometry, thereby determining the identity of the target nucleotide.
A process for detecting the absence or presence of a mutation in a target nucleic acid sequence can be performed by hybridizing a nucleic acid molecule containing the target nucleic acid sequence with at least one primer, which has 3xe2x80x2 terminal base complementarity to the target nucleic acid sequence, to produce a hybridized product; contacting the hybridized product with an appropriate polymerase enzyme and sequentially with one of the four nucleoside triphosphates, then preparing a composition containing the reaction product and a liquid matrix, which absorbs infrared radiation; and detecting the product in the composition by IR-MALDI mass spectrometry, wherein the molecular weight of the product indicates the presence or absence of a mutation next to the 3xe2x80x2 end of the primer in the target nucleic acid molecule. A mutation in a nucleic acid molecule also can be detected, for example, by hybridizing the nucleic acid molecule with an oligonucleotide probe, to produce a hybridized nucleic acid, wherein a mismatch is formed at the site of a mutation; contacting the hybridized nucleic acid with a single strand specific endonuclease, then preparing a composition containing the reaction product and a liquid matrix, which absorbs infrared radiation; and analyzing the composition by IR-MALDI mass spectrometry, wherein the presence of more than one nucleic acid fragment in the composition indicates that the nucleic acid molecule contains a mutation.
A process for identifying the absence or presence of a mutation in a target nucleic acid sequence also can be performed, for example, by performing at least one hybridization on a nucleic acid molecule containing the target nucleic acid sequence with a set of ligation educts and a DNA ligase; preparing a composition containing the reaction product and a liquid matrix, which absorbs infrared radiation; and analyzing the composition by IR-MALDI mass spectrometry. Using such a process, the detection of a ligation product in the composition identifies the absence of a mutation in the target nucleic acid sequence, whereas the detection only of the set of ligation educts in the composition identifies the presence of a mutation in the target nucleic sequence. A process of detecting the presence of a ligation product, as disclosed above, also can be useful for detecting a target nucleotide or a target nucleic acid by performing at least one hybridization on a nucleic acid molecule containing the target nucleotide with a set of ligation educts and a thermostable DNA ligase; preparing a composition containing the reaction product and a liquid matrix, which absorbs infrared radiation; and identifying a ligation product in the composition by IR-MALDI mass spectrometry, thereby detecting the presence of a target nucleotide in the nucleic acid sequence.
Processes for determining a subunit sequence of a biological macromolecule also are provided. A subunit sequence of at least one species of target biological macromolecule, i, can be determined, for example, by contacting the species of target biological macromolecule with one or more agents sufficient to cleave each bond involved in the formation of the target biological macromolecule, to produce a set of nested biological macromolecule fragments, then preparing a composition containing at least one biological macromolecule fragment of the set and a liquid matrix, which absorbs infrared radiation; and determining the molecular mass of the at least one biological macromolecule fragment by IR-MALDI mass spectrometry; and repeating these steps until the molecular mass of each biological macromolecule fragment in the set has been determined, thereby determining the subunit sequence of the species of target biological macromolecule. Such a process is particularly suitable for multiplex analysis of a plurality of i+1 species of target biological macromolecules, wherein each species of target biological macromolecule is differentially mass modified such that a biological macromolecule fragment of each species of target biological macromolecule can be distinguished from a biological macromolecule of each different species by IR-MALDI mass spectrometry.
Processes for determining the nucleotide sequence of at least one species of nucleic acid are provided. Such a process can be performed by synthesizing complementary nucleic acids, which are complementary to the species of nucleic acid to be sequenced, starting from an oligonucleotide primer and in the presence of chain terminating nucleoside triphosphates, to produce four sets of base-specifically terminated complementary polynucleotide fragments; preparing a composition for IR-MALDI, containing the four sets of polynucleotide fragments and a liquid matrix, which absorbs infrared radiation; determining the molecular weight value of each polynucleotide fragment by IR-MALDI mass spectrometry; and determining the nucleotide sequence of the species of nucleic acid by aligning the molecular weight values according to molecular weight. Such a process is particularly suitable to multiplex analysis of a plurality of i+1 species of nucleic acids, which can be sequenced concurrently using i+1 primers, wherein one of the i+1 primers is an unmodified primer or a mass modified primer and the other i primers are mass modified primers, and wherein each of the i+1 primers can be distinguished from the other by IR-MALDI mass spectrometry.
A sequence of a target nucleic acid also can be determined by hybridizing at least one partially single stranded target nucleic acid to one or more nucleic acid probes, each probe containing a double stranded portion, a single stranded portion, and a determinable variable sequence within the single stranded portion, to produce at least one hybridized target nucleic acid, then preparing a composition containing the hybridized target nucleic acid and a liquid matrix, which absorbs infrared radiation; and determining a sequence of the hybridized target nucleic acid by IR-MALDI mass spectrometry based on the determinable variable sequence of the probe to which the target nucleic acid hybridized. If desired, the steps of the process can be repeated a sufficient number of times to determine an entire sequence of a target nucleic acid and, where a plurality of target nucleic acids are to be sequenced, the one or more nucleic acid probes can be immobilized in an array. If desired, the hybridized target nucleic acid can be ligated to the determinable variable sequence prior to preparing the composition for IR-MALDI.
A process for determining the sequence of a target biological macromolecule also can be performed by generating at least two biological macromolecule fragments from the target biological macromolecule, then preparing a composition containing the biological macromolecule fragments and a liquid matrix, which absorbs infrared radiation; and analyzing the biological macromolecule fragments in the composition by IR-MALDI mass spectrometry, thereby determining the sequence of the target nucleic acid molecule. Such a process is particularly useful for ordering two or more portions of a biological macromolecule sequence within a larger sequence.
Also, provided are compositions for IR-MALDI that contain a liquid matrix, which absorbs infrared radiation, and a biological macromolecule. In particular, the biological macromolecule and the liquid matrix are present in a ratio of about 10xe2x88x924 to 10xe2x88x929 biological macromolecule to liquid matrix in the composition. Also provided are these compositions in which the biological macromolecule is present in an amount less than about picomoles of biological macromolecule, preferably about 100 attomoles to about 1 picomole of biological macromolecule. The compositions can further include an additive that facilitates detection of the nucleic acid by IR-MALDI. Supports (or substrates) on which the compositions are deposited are provided.